The long-range goal of our laboratory is to describe meaningful elements of the mechanotransduction process in endothelial cells to further understand the role of hemodynamics in atherosclerosis. Our proposal will test the specific idea that caveolae are sites for the formation of a mechanosensory signaling complex which serves to coordinate biomechanical inputs and induce signaling events that modify endothelial cell structure and function. The working hypothesis is that shear stress-induced mechanotransduction involves a caveolae-based mechanosensory signaling complex that forms when mechanically sensitive integrins translocate to caveolae. We will test this hypothesis by using subcellular fractionation and immunoelectron microscopic techniques to track the distribution of activated integrins following shear stress. We hypothesize that the functional significance of integrin transposition to caveolae is to allow integrins to association with downstream signaling partners. Specifically, integrins associate with a caveolae-based phospho-caveolin/Src/Csk complex which governs RhoA activity and reorganization of the actin cytoskeleton and inhibition of cell cycle in response to shear stress. To test this hypothesis, endothelial cell cultures will be pretreated with integrin inhibiting antibodies, Csk siRNA and caveolin-1 phospho-peptide prior to shear stress. Additionally, specific reduction of caveolae through siRNA knockdown of caveolin-1 and cholesterol sequestering compounds will be used to evaluate the role of rafts/caveolae in integrin-mediated mechanotransduction. In vitro results will be validated in caveolin-1 null mice. Integrins also regulate shear-induced NO production. However, our understanding of how integrin-mediated mechanotransduction processes elicit specific cellular responses to fluid mechanical forces is unclear. We propose that signal specificity is conferred through integrin association with caveolae domains containing specific subsets of signaling molecules which are segregated between focal contacts and the luminal cell surface. Here, we will examine eNOS activity at the luminal surface and within purified caveolae in endothelial cells pretreated with integrin blocking antibodies. We propose that integrins expressed on the luminal endothelial cell surface translocate to caveolae following shear stress where they interact with a SFK/caveolin/PI3-Kinase/Akt complex to activate eNOS. PUBLIC HEALTH RELEVANCE The development of plaques within a blood vessel can impede normal blood flow to organs such as the heart and brain and lead to heart attack or stroke, respectively. Interestingly, atherosclerotic plaques are most often found where blood vessels bifurcate. The flow of blood within these regions is chaotic and turbulent, which alters the normal functioning of the cells which line the blood vessel wall, called endothelial cells. The long-range goal of our research is to understand the role of hemodynamics in atherosclerosis and describe meaningful elements of the mechanotransduction process in endothelial cells. The rationale that underlies this research is that, once a clear understanding of the mechanotransduction pathways is achieved, relevant components may be targeted to attenuate plaque formation in hemodynamic sensitive areas of the vasculature.